CN117378598A - Oocyte cryopreservation liquid and preparation method thereof - Google Patents
Oocyte cryopreservation liquid and preparation method thereof Download PDFInfo
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- CN117378598A CN117378598A CN202311681430.XA CN202311681430A CN117378598A CN 117378598 A CN117378598 A CN 117378598A CN 202311681430 A CN202311681430 A CN 202311681430A CN 117378598 A CN117378598 A CN 117378598A
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- oocyte
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- hydroxyapatite
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- 210000000287 oocyte Anatomy 0.000 title claims abstract description 94
- 238000002360 preparation method Methods 0.000 title claims abstract description 38
- 238000005138 cryopreservation Methods 0.000 title claims abstract description 37
- 239000007788 liquid Substances 0.000 title claims abstract description 34
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims abstract description 51
- 239000000243 solution Substances 0.000 claims abstract description 40
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims abstract description 31
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000007995 HEPES buffer Substances 0.000 claims abstract description 30
- 229930182830 galactose Natural products 0.000 claims abstract description 26
- 229920002134 Carboxymethyl cellulose Polymers 0.000 claims abstract description 12
- 239000001768 carboxy methyl cellulose Substances 0.000 claims abstract description 12
- 235000010948 carboxy methyl cellulose Nutrition 0.000 claims abstract description 12
- 239000008112 carboxymethyl-cellulose Substances 0.000 claims abstract description 12
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 9
- 239000002253 acid Substances 0.000 claims abstract description 8
- 102000007562 Serum Albumin Human genes 0.000 claims abstract description 6
- 108010071390 Serum Albumin Proteins 0.000 claims abstract description 6
- 150000005846 sugar alcohols Polymers 0.000 claims abstract description 6
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 76
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 36
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 28
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 28
- 238000007710 freezing Methods 0.000 claims description 26
- 230000008014 freezing Effects 0.000 claims description 26
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 22
- 239000011550 stock solution Substances 0.000 claims description 20
- 239000002202 Polyethylene glycol Substances 0.000 claims description 19
- 229920001223 polyethylene glycol Polymers 0.000 claims description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- 229920005862 polyol Polymers 0.000 claims description 9
- 150000003077 polyols Chemical class 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 8
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 4
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 3
- 230000007062 hydrolysis Effects 0.000 claims description 3
- 238000006460 hydrolysis reaction Methods 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 239000007790 solid phase Substances 0.000 claims description 3
- PUPZLCDOIYMWBV-UHFFFAOYSA-N (+/-)-1,3-Butanediol Chemical compound CC(O)CCO PUPZLCDOIYMWBV-UHFFFAOYSA-N 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 6
- 230000002195 synergetic effect Effects 0.000 abstract description 5
- 230000004071 biological effect Effects 0.000 abstract description 3
- 102000008100 Human Serum Albumin Human genes 0.000 description 22
- 108091006905 Human Serum Albumin Proteins 0.000 description 22
- 210000004027 cell Anatomy 0.000 description 19
- 239000008213 purified water Substances 0.000 description 17
- 230000035699 permeability Effects 0.000 description 14
- 238000004017 vitrification Methods 0.000 description 14
- 239000002577 cryoprotective agent Substances 0.000 description 12
- 230000000052 comparative effect Effects 0.000 description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 230000008569 process Effects 0.000 description 9
- 238000001816 cooling Methods 0.000 description 8
- 239000013078 crystal Substances 0.000 description 8
- 239000011734 sodium Substances 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 6
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 239000003223 protective agent Substances 0.000 description 6
- 210000000625 blastula Anatomy 0.000 description 5
- 210000000170 cell membrane Anatomy 0.000 description 5
- 230000006378 damage Effects 0.000 description 5
- 150000002016 disaccharides Chemical class 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 150000002632 lipids Chemical class 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 230000003204 osmotic effect Effects 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 239000005639 Lauric acid Substances 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000002438 mitochondrial effect Effects 0.000 description 3
- 150000002772 monosaccharides Chemical class 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical group OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 231100000045 chemical toxicity Toxicity 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 2
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000036542 oxidative stress Effects 0.000 description 2
- 125000000914 phenoxymethylpenicillanyl group Chemical group CC1(S[C@H]2N([C@H]1C(=O)*)C([C@H]2NC(COC2=CC=CC=C2)=O)=O)C 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- WQZGKKKJIJFFOK-DHVFOXMCSA-N L-galactose Chemical compound OC[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O WQZGKKKJIJFFOK-DHVFOXMCSA-N 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 229920006321 anionic cellulose Polymers 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000007420 reactivation Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- XRZSXZKAAVJHQX-UHFFFAOYSA-M sodium;ethanesulfonate;2-piperazin-1-ylethanol Chemical compound [Na+].CCS([O-])(=O)=O.OCCN1CCNCC1 XRZSXZKAAVJHQX-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Abstract
The invention relates to an oocyte cryopreservation liquid and a preparation method thereof, wherein the oocyte cryopreservation liquid comprises serum albumin, galactose, polyalcohol, PVA, modified nano hydroxyapatite, HEPES buffer solution and the like. Wherein, the modified nano-hydroxyapatite is modified by hydroxyapatite grafted alkyl acid and carboxymethyl cellulose, so that the biological activity and dispersibility of the nano-hydroxyapatite are effectively improved; galactose and polyalcohol are compounded to produce good synergistic effect with the modified nano hydroxyapatite. Compared with the prior art, the oocyte cryopreservation solution scheme is effectively optimized, has obvious lifting effect and good application prospect, and provides reference and reference for the oocyte cryopreservation technology.
Description
Technical Field
The invention belongs to the technical field of assisted reproduction, and particularly relates to oocyte cryopreservation liquid and a preparation method thereof.
Background
Cryopreservation techniques have become one of the indispensable preservation methods in the biomedical field. The technology can store biological materials such as cells, tissues, organs and the like in an ultralow temperature environment, so that the metabolism of the cells is greatly slowed down or even stopped, and the cells can continue to develop once the normal physiological temperature is restored. The most common cryopreservation method at present is vitrification cryopreservation, which is a method of combining rapid freezing with a cryoprotectant, replacing intracellular moisture with the cryoprotectant with high concentration, and rapidly cooling to convert the cells from a liquid state into an amorphous solid state with a glass-like shape. In the vitrification cryopreservation process, the proper cryoprotectant can reduce the change or loss of cell characteristics, and plays a role in protecting cells.
The oocyte cryopreservation technology is a vitrification cryopreservation method for preserving oocytes, and is widely applied to fertility preservation and assisted reproduction technologies. The oocyte freezing solution is used as a freezing protective agent, is one of the most critical basic substances in the oocyte freezing technology, and aims to avoid the inactivation of oocytes caused by the formation of ice crystals in a freezing system. However, the high concentration permeability cryoprotectants (such as DMSO and the like) used in the current cryoprotectants are easy to cause chemical toxicity, and once the cryoprotectants are operated too slowly, strong permeability damage can be caused, so that the skeleton structure of the oocyte is broken; the vitrification process can also cause changes in the lipid content of the oocyte, affecting the integrity of the oocyte membrane and, in turn, the subsequent fertilization and development processes. Moreover, existing cryoprotectants are not effective in controlling ice crystal growth, thereby damaging the cells.
The prior art, for example, discloses a cryopreservation solution and a preparation method thereof (CN 111789108B), which comprises PVA, glycol and sucrose, wherein PVA is used as a main low-temperature protective agent component, but a DMSO permeability protective agent is still needed, and the quality change in the vitrification process of oocytes is not improved obviously.
Therefore, it is needed to provide a new technical scheme based on the prior art, and develop oocyte cryopreservation liquid with lower cytotoxicity and higher efficiency for inhibiting lipid change in the cryopreservation process.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides an oocyte frozen stock solution and a preparation method thereof.
The invention aims at providing an oocyte cryopreservation liquid, which comprises the following components in parts by weight:
serum albumin 0.1-5 parts
Galactose 9-20 parts
Polyethylene glycol 1-8 parts
10-30 parts of polyol
PVA 1-6 parts
0.01-0.1 part of modified nano hydroxyapatite
HEPES buffer solution 20-50 parts
20-50 parts of water;
wherein,
the modified nano-hydroxyapatite is a product obtained by grafting alkyl acid and carboxymethyl cellulose with nano-hydroxyapatite.
During freezing, oocytes may be damaged by oxidative stress, resulting in a decrease in their activity and reactivation rate. Maintaining the mitochondrial activity of oocytes can promote intracellular metabolism and energy production, helping to reduce this damage. The nano hydroxyapatite can reduce the generation of active oxygen in the mitochondria of the oocyte, promote the proliferation and differentiation of cells, thereby protecting the oocyte from being damaged by oxidative stress and reducing injury and apoptosis.
PVA can effectively inhibit the generation of ice crystals in the vitrification process and improve the vitrification capacity of the refrigerating fluid.
PVA solution is used as an impermeable cryoprotectant to protect the integrity of cells by maintaining the osmotic pressure of cells at low temperature, reducing the formation of ice crystals in the cells, inhibiting protein denaturation, promoting cell resuscitation and other various functions. The research shows that the survival rate of the oocyte after the common vitrification freezing solution is frozen is about 80 percent, and the survival rate of the frozen oocyte after the PVA is added is frozen is more than 95 percent.
Polyols act as osmotic cryoprotectants, preventing ice crystal formation and disrupting cellular structure, primarily by lowering the intracellular freezing point. Ice crystal formation damages the structure and function of the cell, while the protection of the polyol is to inhibit ice crystal formation, thereby protecting the integrity of the cell.
Galactose and polyalcohol are compounded to produce synergistic effect, and the stability of lipid is induced by preventing the polymerization of intracellular proteins, so that the blastula rate of the oocyte after vitrification is improved.
Polyethylene glycol, preferably PEG8000, is nontoxic, and researches prove that the polyethylene glycol can improve the normal rate of oocyte after vitrification, can prevent degradation of some lecithins, and can effectively maintain the integrity of cell membranes.
Ethylene glycol acts as a permeability protectant, with the particular permeability protectant providing effective protection based on their membrane permeability and other characteristics, as well as the maturation stage of the oocyte. Researches show that the permeability of ethylene glycol is half that of PG and DMSO, but the toxicity is low, and the ethylene glycol is used as a first choice of permeability protective agent, and in the vitrification process, hydrogen bonds are formed between the penetrating plasma membrane and water molecules to reduce the formation of ice crystals, while just PEG8000 can promote the permeability of cell membranes, and the two can compensate each other, and meanwhile, the PEG8000 not only can improve the permeability of the membranes, but also can prevent the cells from swelling and breaking due to the water flowing into the cells when the cell membranes are damaged in the vitrification process.
Galactose, which is a monosaccharide, has a smaller partial molar volume than disaccharide, has weaker capacity to displace water than disaccharide, can reduce the concentration of the intracellular and extracellular permeability protective agent, and can provide optimal low-temperature storage conditions; in addition, the sugar interacts with the cell bilayer to prevent the polymerization of intracellular proteins and induce the stability of lipid; the biocompatibility with glycol is better.
The osmotic pressure of monosaccharide and disaccharide with the same concentration is similar, but the viscosity of disaccharide solution is high, galactose solution can be better mixed with other permeability protective agents, in addition, research shows that oocyte has higher tolerance to monosaccharide solution than disaccharide solution, and can better protect cells from damage.
Further, the serum albumin may be selected from human serum albumin or a substitute, such as any one of sodium dodecyl sulfate, SDS, or hydroxypropyl cellulose, HPC.
Further, the PVA has a degree of hydrolysis of greater than 80%.
Further, the polyol is selected from propylene glycol, ethylene glycol or butylene glycol.
Further, the polyethylene glycol has a molecular weight of 4000-8000.
Further, the galactose is selected from D-galactose or L-galactose.
Further, the concentration of the HEPES buffer solution is 50-70 wt%.
Further, the buffer is selected from any one of HEPES buffer and M199 buffer.
The invention also provides a preparation method of the oocyte frozen stock solution, which comprises the following steps:
(1) Preparation of modified nano hydroxyapatite: adding nano hydroxyapatite, alkyl acid and carboxymethyl cellulose into an ethanol water solution in a reactor provided with a condensing device, heating and refluxing, then removing a solvent, washing a solid phase with water, and drying to obtain the modified nano hydroxyapatite;
(2) Preparation of oocyte cryopreservation liquid: and (3) blending the modified nano hydroxyapatite, galactose, polyethylene glycol, polyalcohol, PVA and water, and adding serum albumin and HEPES buffer solution after stirring to obtain the oocyte frozen stock solution.
Further, the mass ratio of the nano-hydroxyapatite to the alkyl acid to the carboxymethyl cellulose is 1:5:5-1:10:10.
Further, in the aqueous solution of ethanol, the concentration of ethanol is 50-70 wt%.
Further, the heating temperature is 80-100 ℃.
The invention has the following beneficial effects:
1. the oocyte freezing solution provided by the invention is compounded by components such as PVA, polyalcohol, galactose and the like, so that a good synergistic effect is generated. The chemical toxicity of the vitrification reagent is effectively reduced, the change of lipid in the vitrification process of the oocyte is controlled, the blastula rate, the survival rate and the development capability of the vitrified oocyte are improved, and the quality of the vitrified oocyte is improved.
2. The modified nano-hydroxyapatite is prepared from nano-hydroxyapatite and alkyl acid grafted carboxymethyl cellulose, has biological effects of preventing recrystallization, increasing the mitochondrial activity of oocytes and reducing active oxygen, has a long-chain alkyl structure grafted on the surface, can be used as an energy metabolite of cells while improving the dispersibility of the nano-hydroxyapatite, has immune enhancement and antibacterial effects, is combined with the anti-blocking and water-retaining effects of the carboxymethyl cellulose, comprehensively improves the biocompatibility and the dispersibility of the material, and improves the application effect of the nano-hydroxyapatite in oocyte frozen stock.
3. Compared with the prior art, the method has the advantages that the oocyte cryopreservation solution scheme is effectively optimized, the survival rate of the unfrozen cells is greatly improved, the method has good application prospect, and reference are provided for the oocyte cryopreservation technology.
Drawings
FIG. 1 is an enlarged view of an oocyte in example 1 of the present invention.
FIG. 2 is an enlarged view of blastula in example 1 of the present invention.
Detailed Description
The present invention is described in detail below with reference to examples to facilitate understanding of the present invention by those skilled in the art. It is specifically pointed out that the examples are given solely for the purpose of illustration of the invention and are not to be construed as limiting the scope of the invention, since numerous insubstantial modifications and variations of the invention will be within the scope of the invention, as described above, will become apparent to those skilled in the art. Meanwhile, the raw materials mentioned below are not specified, and are all commercial products; the process steps or preparation methods not mentioned in detail are those known to the person skilled in the art.
In this example, all formulation and testing occurred at 25 ℃ unless otherwise indicated. The following ingredients will be used in the examples and have the indicated functions.
Oocyte: is taken from a C57BL/6 female mouse of Zhuhai Baitong biotechnology Co., ltd;
human serum albumin: plays roles in maintaining osmotic pressure, buffering and nutrition in cell culture, and is purchased from Shenzhen Wei Guang biological products, inc.;
galactose: has synergistic effect with polyol permeability cryoprotectant, purchased from Sigma under the trade name G5388;
ethylene glycol: a polyol, a permeable cryoprotectant, purchased from Sigma under the designation 102466;
polyethylene glycol: PEG8000, a high molecular polymer, can promote the permeability of cell membranes, and is purchased from Sigma with the brand number of 89510;
PVA: polyvinyl alcohol, impermeable cryoprotectant, purchased from Sigma under the trade name P8136, degree of hydrolysis 88%;
HEPES: 4-hydroxyethyl piperazine ethanesulfonic acid, a zwitterionic buffer, used to maintain a constant pH during cell culture, purchased from Sigma under the designation H4034; HEPES buffer concentration was 50 wt%;
HEPES-Na: 4-hydroxyethyl piperazine ethanesulfonic acid sodium salt, purchased from Sigma;
modified nano hydroxyapatite: is used for increasing the mitochondrial activity of the oocyte system, reducing the generation of active oxygen and self-making. Nano hydroxyapatite raw material is purchased from Sigma with the brand 919101;
lauric acid: a saturated fatty acid is a raw material of modified nano hydroxyapatite, purchased from Sigma with the trade name of W261416;
carboxymethyl cellulose: an anionic cellulose ether is a raw material of modified nano hydroxyapatite, purchased from Sigma with the brand number of C5678.
Example 1
An oocyte cryopreservation solution A comprises the following components in parts by weight:
human serum albumin 5 parts
Galactose 9 parts
Polyethylene glycol 2 parts
15 parts of ethylene glycol
PVA 1 part
0.05 part of modified nano hydroxyapatite
44.95 parts of HEPES buffer
23 parts of purified water;
in addition, the invention discloses a freezing balance liquid A, which comprises the following components in parts by weight:
human serum albumin 10 parts
7.5 parts of ethylene glycol
PVA 1.5 parts
HEPES buffer 46 parts
Purified water was 35 parts.
The preparation method of the oocyte cryopreservation solution comprises the following steps:
(1) Preparation of modified nano hydroxyapatite:
immersing nano hydroxyapatite, lauric acid and carboxymethyl cellulose (1:5:5, m/m/m) into a sufficient amount of ethanol water solution (65 wt%), stirring for 15 min at room temperature, transferring to a water bath heater, heating to 85 ℃, stirring and refluxing for reaction 3 h, cooling to room temperature, transferring to a filtering centrifuge, removing water and ethanol, cleaning the residual solid phase substance with purified water, and drying at 60 ℃ in a constant temperature drying oven for 5 h to obtain the modified nano hydroxyapatite;
(2) Preparation of oocyte cryopreservation liquid:
mixing purified water, PVA, polyethylene glycol, ethylene glycol, galactose and the modified nano-hydroxyapatite according to the parts by weight, stirring for 1 h at 80 ℃, cooling to room temperature, adding human serum albumin and HEPES buffer solution, and regulating the pH value to 7.2 by using HEPES-Na to obtain oocyte frozen stock solution A;
the preparation method of the freezing balance liquid comprises the following steps:
mixing the purified water, PVA and ethylene glycol according to the parts by weight, stirring for 1 h at 80 ℃, cooling to room temperature, adding human serum albumin and HEPES buffer solution, and regulating the pH to 7.2 by using HEPES-Na to obtain the frozen equilibrium liquid A.
Example 2
An oocyte cryopreservation liquid B comprises the following components in parts by weight:
human serum albumin 5 parts
Galactose 10 parts
Polyethylene glycol 5 parts
15 parts of ethylene glycol
PVA 1 part
0.04 part of modified nano hydroxyapatite
HEPES buffer 39.96 parts
24 parts of purified water;
in addition, the invention discloses a freezing balance liquid B, which comprises the following components in parts by weight:
human serum albumin 15 parts
15 parts of ethylene glycol
70 parts of HEPES buffer.
The preparation method of the oocyte cryopreservation solution comprises the following steps:
(1) The preparation method of the modified nano hydroxyapatite is the same as in example 1;
(2) Preparation of oocyte cryopreservation liquid:
mixing purified water, PVA, polyethylene glycol, ethylene glycol, galactose and the modified nano-hydroxyapatite according to the parts by weight, stirring for 1 h at 80 ℃, cooling to room temperature, adding human serum albumin and HEPES buffer solution, and regulating the pH value to 7.2 by using HEPES-Na to obtain oocyte frozen stock solution B;
the preparation method of the freezing balance liquid comprises the following steps:
according to the parts by weight, adding ethylene glycol and human serum albumin into HEPES buffer, and regulating the pH to 7.2 by using HEPES-Na to obtain the frozen equilibrium liquid B.
Example 3
An oocyte cryopreservation liquid C comprises the following components in parts by weight:
human serum albumin 1 part
Galactose 10 parts
Polyethylene glycol 5 parts
15 parts of ethylene glycol
PVA 1 part
0.06 part of modified nano hydroxyapatite
HEPES buffer 38 parts
29.94 parts of purified water;
a freezing balance liquid A comprises the following components in parts by weight:
human serum albumin 10 parts
7.5 parts of ethylene glycol
PVA 1.5 parts
HEPES buffer 46 parts
Purified water was 35 parts.
The preparation method of the oocyte cryopreservation solution comprises the following steps:
(1) The preparation method of the modified nano hydroxyapatite is the same as in example 1;
(2) Preparation of oocyte cryopreservation liquid:
mixing purified water, PVA, polyethylene glycol, ethylene glycol, galactose and the modified nano-hydroxyapatite according to the parts by weight, stirring for 1 h at 80 ℃, cooling to room temperature, adding human serum albumin and HEPES buffer solution, and regulating the pH value to 7.2 by using HEPES-Na to obtain oocyte frozen stock solution C;
the preparation method of the frozen equilibrium liquid A is the same as in example 1.
Example 4
An oocyte cryopreservation solution D comprises the following components in parts by weight:
human serum albumin 1 part
Galactose 15 parts
Polyethylene glycol 5 parts
20 parts of ethylene glycol
PVA 2 parts
0.08 portion of modified nano hydroxyapatite
HEPES buffer 36.92 parts
20 parts of purified water;
a freezing balance liquid B comprises the following components in parts by weight:
human serum albumin 15 parts
15 parts of ethylene glycol
70 parts of HEPES buffer.
The preparation method of the oocyte cryopreservation solution comprises the following steps:
(1) The preparation method of the modified nano hydroxyapatite is the same as in example 1;
(2) Preparation of oocyte cryopreservation liquid:
mixing purified water, PVA, polyethylene glycol, ethylene glycol, galactose and the modified nano-hydroxyapatite according to the parts by weight, stirring for 1 h at 80 ℃, cooling to room temperature, adding human serum albumin and HEPES buffer solution, and regulating the pH value to 7.2 by using HEPES-Na to obtain oocyte frozen stock solution D;
the preparation method of the frozen equilibrium liquid B is the same as in example 2.
Example 5
An oocyte cryopreservation solution E comprises the following components in parts by mass:
human serum albumin 2 parts
Galactose 12 parts
Polyethylene glycol 5 parts
20 parts of ethylene glycol
PVA 1.5 parts
0.08 portion of modified nano hydroxyapatite
29.42 parts of HEPES buffer
30 parts of purified water;
a freezing balance liquid A comprises the following components in parts by weight:
human serum albumin 10 parts
7.5 parts of ethylene glycol
PVA 1.5 parts
HEPES buffer 46 parts
Purified water was 35 parts.
The preparation method of the oocyte cryopreservation solution comprises the following steps:
(1) The preparation method of the modified nano hydroxyapatite is the same as in example 1;
(2) Preparation of oocyte cryopreservation liquid:
mixing purified water, PVA, polyethylene glycol, ethylene glycol, galactose and the modified nano-hydroxyapatite according to the parts by weight, stirring for 1 h at 80 ℃, cooling to room temperature, adding human serum albumin and HEPES buffer solution, and regulating the pH value to 7.2 by using HEPES-Na to obtain oocyte frozen stock solution E;
the preparation method of the frozen equilibrium liquid A is the same as in example 1.
Comparative example 1
The difference between the oocyte frozen stock solution F in this comparative example and the example 1 is that the oocyte frozen stock solution F in this comparative example does not contain lauric acid and carboxymethyl cellulose grafted modified nano-hydroxyapatite, but is replaced by the unmodified nano-hydroxyapatite with equal mass fraction, and other components, mass fraction and preparation method are the same as in example 1.
A freezing balance liquid A, each component and preparation method are the same as in example 1.
Comparative example 2
The difference between the present comparative example and example 2 is that the present comparative example was conducted in such a manner that the oocyte frozen stock G was not added with ethylene glycol but replaced with purified water of equal parts by mass, and the other components, parts by mass and preparation method were the same as in example 2.
A freezing balance liquid B, each component and the preparation method are the same as in example 2.
Comparative example 3
The difference between the present comparative example and example 3 is that galactose was not added to the oocyte frozen stock solution H in the present comparative example, but purified water was used instead of the same parts by mass, and other components, parts by mass and preparation methods were the same as those of example 3.
A freezing balance liquid A, each component and preparation method are the same as in example 1.
Test case
Oocytes were tested for biological activity in examples 1-5 and comparative examples 1-3.
The test method is as follows:
s1, freezing: respectively adding oocytes into the freezing balance solutions of the examples 1-5 and the comparative examples 1-3 for balancing for 2.5 min, transferring the oocytes into the corresponding oocyte freezing solutions A-H for 20-s, transferring the oocytes onto a freezing carrier rod, and rapidly putting into liquid nitrogen for freezing and preserving;
s2, thawing: the oocytes are placed in a resuscitation fluid at 37 ℃ for 1 min to be balanced, then transferred into a dilution fluid for 3 min, and finally transferred into a cleaning fluid for 5 min. After thawing, transferring the oocyte into a proper culture medium for incubation until complete recovery, calculating the survival rate of the oocyte, then injecting and culturing single sperm in the cytoplasm, and calculating the fertilization rate and blastula rate of the oocyte.
Wherein the resuscitation fluid consists of 1 part of human serum albumin, 20 parts of galactose and 79 parts of HEPES buffer; the diluent consists of 5 parts of human serum albumin, 10 parts of galactose and 75 parts of HEPES buffer solution; the cleaning solution consists of 5 parts of human serum albumin and 95 parts of HEPES buffer.
The results obtained are shown in Table 1 and FIGS. 1 and 2.
Table 1 test results
FIG. 1 is an enlarged view of an oocyte in example 1 of the present invention.
FIG. 2 is an enlarged view of blastula in example 1 of the present invention.
As can be seen from Table 1, the oocyte frozen stock solutions of examples 1 to 5 all have excellent bioactivity and stability. In practical application test, the oocyte activity of the example 1 is obviously better than that of the comparison example 1 without modified nano-hydroxyapatite, and the oocyte activity of the examples 2 and 3 is also obviously better than that of the comparison examples 2 and 3. Proved by the technical scheme of the invention, the modified nano hydroxyapatite, the polyol and the galactose can generate a synergistic effect, so that the quality of the thawed oocyte can be effectively improved and the development capacity of the vitrified oocyte can be improved while the stability of the frozen stock is ensured.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (9)
1. The oocyte cryopreservation liquid is characterized by comprising the following components in parts by mass:
serum albumin 0.1-5 parts
Galactose 9-20 parts
Polyethylene glycol 1-8 parts
10-30 parts of polyol
PVA 1-6 parts
0.01-0.1 part of modified nano hydroxyapatite
HEPES buffer solution 20-50 parts
20-50 parts of water;
wherein,
the modified nano-hydroxyapatite is a product obtained by grafting alkyl acid and carboxymethyl cellulose with nano-hydroxyapatite.
2. The oocyte frozen stock solution according to claim 1, characterized in that the degree of hydrolysis of PVA is more than 80%.
3. The oocyte frozen stock solution according to claim 1, characterized in that the polyol is selected from propylene glycol, ethylene glycol or butylene glycol.
4. The oocyte frozen stock solution according to claim 1, characterized in that the polyethylene glycol has a molecular weight of 4000-8000.
5. The oocyte frozen stock solution according to claim 1, characterized in that the concentration of HEPES buffer is 50-70 wt%.
6. The method for preparing the oocyte frozen stock solution according to any one of claims 1 to 5, wherein the method for preparing the oocyte frozen stock solution comprises the following steps:
(1) Preparation of modified nano hydroxyapatite: adding nano hydroxyapatite, alkyl acid and carboxymethyl cellulose into an ethanol water solution in a reactor provided with a condensing device, heating and refluxing, then removing a solvent, washing a solid phase with water, and drying to obtain modified nano hydroxyapatite;
(2) Preparation of oocyte cryopreservation liquid: and (3) blending the modified nano hydroxyapatite, galactose, polyethylene glycol, polyalcohol, PVA and water, and adding serum albumin and HEPES buffer solution after stirring to obtain the oocyte frozen stock solution.
7. The method for preparing the oocyte frozen stock solution according to claim 6, wherein the mass ratio of the nano-hydroxyapatite to the alkyl acid to the carboxymethyl cellulose is 1:5:5-1:10:10.
8. The method for preparing an oocyte freezing solution according to claim 6, wherein the concentration of ethanol in the aqueous solution of ethanol is 50-70 wt%.
9. The method for preparing an oocyte freezing solution according to claim 6, wherein the heating temperature is 80-100 ℃.
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